Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of receptor-coupled ion channels (Karlin, 2002; Kellar and Xiao, 2007; Lester et al., 2004; Sine and Engel, 2006). The superfamily includes, for example, muscle nAChRs, neuronal nAChRs, 5-hydroxytryptamine type 3 (5-HT3) receptors, γ-aminobutyric acid type A (GABAA) and GABAC receptors and glycine receptors. Neuronal nAChRs are differentially expressed in many regions of the central nervous system (CNS) and peripheral nervous system (PNS) (Alkondon and Albuquerque, 2004; Kellar et al., 1999; Le Novere et al., 2002; Lukas et al., 1999; Paterson and Nordberg, 2000; Skok, 2002).
Neuronal nAChRs are fundamental to many physiological functions of the CNS and PNS. In brain, nAChRs modulate the release of major CNS neurotransmitters, including dopamine (DA), norepinephrine, acetylcholine (ACh), γ-aminobutyric acid (GABA) and glutamate, which are associated with arousal, reward, mood and affect, attention, learning and memory (Dani and Bertrand, 2007; Levin et al., 2006; Wonnacott et al., 2006). In PNS, neuronal nAChRs mediate fast neurotransmission provided by ACh at virtually all autonomic ganglia, sensory ganglia and adrenal gland (De Biasi, 2002; Wang et al., 2002).
These receptors are implicated in many pathological conditions and processes, including aging, addiction, pain, obesity, schizophrenia, epilepsy, mania and manic depression, anxiety, Alzheimer's disease, learning deficit, cognition deficit, attention deficit, memory loss, Lewy Body Dementia, Attention Deficit Hyperactivity Disorder, Parkinson's disease, Huntington's disease, Tourette's syndrome, amyotrophic lateral sclerosis, inflammation, stoke and spinal-cord injury (Gotti and Clementi, 2004; Lindstrom, 1997).
nAChRs also mediate the pharmacological effects of exogenous nicotinic ligands, such as nicotine, lobeline, hexamethonium and mecamylamine (Daly, 2005; Jensen et al., 2005). Moreover, these receptors mediate the effects of nicotine, the addictive agent in tobacco (Laviolette and van der Kooy, 2004; Mansvelder and McGehee, 2002) and the main reason that more than one billion people worldwide smoke.
Neuronal nAChRs are pentameric membrane proteins composed of five subunits. To date, nine α subunits (α2-α10) and three β subunits (β2-(β4) have been found in vertebrates (Le Novere et al., 2002; Lindstrom, 1996; Millar, 2003). Different combinations of these α and β subunits define nAChR subtypes. Although the theoretical number of potential subtypes is very large, a much smaller number of native nAChR subtypes that represent the majority of neuronal nAChRs, including two heteromeric subtypes, α4β2 and α3β4, and one homomeric subtype, α7. In most areas of mammalian brain, the α4β2 subtype represents the predominant population of nAChRs; in ganglia, however, α3β4 nAChRs are the major subtype (Flores et al., 1996; Flores et al., 1992; Gotti et al., 2006; Paterson and Nordberg, 2000; Skok, 2002; Whiting and Lindstrom, 1987).
The nAChRs have been objects on interest for more than 100 years (Langley, 1907). It has been known for many years that ACh, the endogenous agonist, and nicotine, the classical ligand that defines nAChRs, change the functional states of nAChRs. Initially, application of the ligands allows conduction of Na+, K+ and Ca2+ ions, which can lead to membrane depolarization and altered cell function. However, if ACh or nicotine is continuously applied, nAChRs quickly become unresponsive to the presence of the ligands, temporarily stopping conduction of the cations. It is an obviously fortunate property of this agonist-receptor system to cells; otherwise, an agonist may cause the channels to be open too long, which would likely interfere with normal cell functions and eventually may even lead to cell death (Gahring and Rogers, 2005).
Many mechanisms and models have been proposed to explain the complex relationship between nAChRs and their ligands. Though far from complete, the following simplified model is widely accepted by researchers in the field. nAChRs are allosteric proteins that respond to the action of ACh at the binding site by changing the status of the channel gate to carry out the function of the nAChR (Changeux and Edelstein, 1998; Changeux and Edelstein, 2005; Colquhoun and Sakmann, 1998; Hogg and Bertrand, 2007; Sine and Engel, 2006; Unwin, 2005). The receptors have at least three discrete conformational states: a resting state (closed), an open state (opened) and a desensitized state (closed) (Karlin, 1967; Karlin, 2002; Katz and Thesleff, 1957). A particular nicotinic ligand, such as ACh, has a certain affinity for each of the three states. In the absence of bound ligand, nAChRs fluctuate among all three conformational states, but most of the time they are in the resting state. The binding of a ligand to a certain state of the receptor increases the probability of the receptor to be in that state. For example, an agonist binds with a reasonably high affinity to the open state of a receptor, and thus increases the probability of it being in the open (active) conformational state. For a population of receptors, the overall initial effect of the agonist is to shift a certain subpopulation of receptors from the resting state to the open state. In the open state, cations flow through the channel. However, the agonist has an even higher affinity for the desensitized state of the receptor; therefore, the eventual effect of an agonist is to “drive” receptor population from the resting and open states to the desensitized state, in which receptors remain closed. The kinetic rates for transitions between states vary greatly among different nAChR subtypes, which contribute to the great functional diversity of neuronal nAChRs.
In addition to ACh and nicotine, many other natural products and synthetic compounds act on nAChRs (Cassels et al., 2005; Daly, 2005; Jensen et al., 2005; Paterson and Nordberg, 2000). Nicotinic ligands belong to the following three major classes according to their actions.
(1) Agonists. Nicotinic agonists, such as ACh or nicotine, activate nAChRs leading to the opening of their channels, which allows cations to cross the membrane; but prolonged presence of agonists desensitizes the receptors. The actions can be explained by the three-state model, with some speculations. Agonists have low binding affinity at the resting state of nAChRs, higher affinity at the open state, and highest binding affinity at the desensitized state. After an agonist binds, the transition from the resting state to the open state is fast, but the transition from open state to desensitized state is slow. Therefore, agonists can activate receptors to open their channels initially but if present for an extended period, agonists desensitize receptors to close the channels.
(2) Competitive Antagonists. A competitive antagonist, such as dihydro-β-erythroidine (DHβE), does not activate nAChRs but prevents agonists from activating nAChRs by binding to the ACh site. A possible mechanism is that competitive antagonists have higher binding affinity at the resting state of receptors than at the open state; therefore, they do not increase the probability of the open state but can prevent agonists from binding to the ACh site.
(3) Noncompetitive Antagonists. A noncompetitive antagonist, such as mecamylamine, does not activate nAChRs but prevents an agonist from activating nAChRs by binding to a site different from the ACh site. For example, the binding site for mecamylamine is in the central pore of the receptors, and so it blocks the pathway for ions, preventing the function of an agonist (Bertrand et al., 1990; Webster et al., 1999).
More than 100 years ago, nicotine was found to both stimulate and block responses of autonomic ganglia (Langley, 1905; Langley and Dickenson, 1889). The concept of desensitization of muscle nAChRs was proposed 50 year ago (Katz and Thesleff, 1957). Since then, it has been widely accepted that all nicotinic agonists have the dual actions of activation and desensitization of nAChRs and that all muscle and neuronal nAChR subtypes can be desensitized by agonists (Giniatullin et al., 2005; Jensen et al., 2005; Lindstrom, 2002; Quick and Lester, 2002; Wang and Sun, 2005).
It is obvious that the activation of neuronal nAChRs by ACh is important for physiological functions. For example, the activation of postsynaptic nAChRs is essential for conducting fast neurotransmissions by ACh. But it is not very clear if the desensitization of neuronal nAChRs by ACh plays important roles in physiological functions. In fact, the desensitization of neuronal nAChRs should be very brief and scattered, if it occurs, because ACh released from cells is rapidly hydrolyzed by acetylcholinesterase (Kellar, 2006; Zhou et al., 2002).
Pharmacologically, long lasting desensitization of neuronal nAChRs can be produced by applying acetylcholinesterase inhibitors, or by applying agonists that are not rapidly degraded or removed from the receptor vicinity. Nicotine is not readily degraded in vivo (t1/2>1 hour). In a smoker's brain, the nicotine concentration is high enough to desensitize α4β2 nAChRs for prolonged time periods (Brody et al., 2006; Fitch et al., 2003; Ghosheh et al., 2001; Gourley and Benowitz, 1997; Henningfield et al., 1993; Kellar et al., 1999; Kuryatov et al., 2005).
A fundamental question of nicotinic signaling in the CNS is how each of these two opposite actions contributes to the overall pharmacological effects of nicotine. Most studies have focused on the activation of nAChRs to understand nicotine's effects; therefore, there is a rich literature on the activation of nAChRs by nicotine in vitro and in vivo. Since it was conceptualized, there have been concerns that desensitization might be only an experimental phenomenon with minimum physiological or even pharmacological significance (Colquhoun and Sakmann, 1998; Katz and Thesleff, 1957). However, many investigators have recognized that desensitization of the receptors by nicotine plays potentially important roles in nicotine's effects in brain (Gahring and Rogers, 2005; Giniatullin et al., 2005; Kellar et al., 1999; Lu et al., 1999; Quick and Lester, 2002; Wang and Sun, 2005). In rats, for example, a single injection of nicotine initially stimulates prolactin release, but it then blocks subsequent nicotine-stimulated prolactin release for several hours or longer (Hulihan-Giblin et al., 1990; Sharp and Beyer, 1986). To explain this observation, nicotine was proposed to act as a “time-averaged antagonist” of nAChRs (Hulihan-Giblin et al., 1990), meaning that after a brief initial stimulation of brain nAChRs, it causes a long-lasting desensitization of the receptors that prevents their function for an extended period of several hours. This concept, “time-averaged antagonist”, indicted that the desensitization of nAChRs might be the predominant mechanism for some of nicotine's in vivo pharmacological effects.
The brain α4β2 nAChRs are implicated in the addictive effects of nicotine (Flores et al., 1992; Marubio et al., 2003; Maskos et al., 2005; Picciotto et al., 1998; Tapper et al., 2004). It is widely accepted that the mesocorticlimbic dopamine system plays a central role in the rewarding effects of drugs (Nestler, 2005); and like many other addictive drugs, nicotine elevates dopamine in the nucleus accumbens (NAc), which is believed to have a vital role in nicotine addiction. Interestingly, a single injection of nicotine in rats elevates the dopamine concentration in NAc for hours (Di Chiara, 2000; Pidoplichko et al., 2004). It is very difficult to explain this long lasting elevation of dopamine only by activation of nAChRs, which is very brief. Similarly, it is very difficult to explain the lasting pleasure and relief felt by smokers after smoking by only nAChR activation. A complex mechanism has been proposed (Dani and Bertrand, 2007; Dani and Harris, 2005), in which nicotine initially causes activation of α4β2 nAChRs on DA neurons, which originate in the ventral tegmental area (VTA) and project to the NAc, leading to elevate DA in the NAc. Then, when the α4β2 nAChRs on DA neurons are desensitized and thus no longer increasing DA release, the α4β2 nAChRs on inhibitory GABaergic interneurons in the VTA are also desensitized, which decreases the inhibitory influence of GABA on the DA neurons in the VTA. Thus, the lasting desensitization of α4β2 nAChRs on inhibitory GABAergic interneurons produces a sustained overall stimulation of DA release in the NAC.
All nicotinic agonists have these dual actions of activation and desensitization. Therefore, it has been very difficult, if not impossible, to determine conclusively how much each of the two actions contributes to the rewarding effects of nicotine and to addiction. However, it is supported by accumulating experimental evidence that desensitization plays very important roles in mediating in vivo effects of nicotine and other nicotinic agonists.
Native subtypes of nAChRs are differently expressed in many regions of the CNS and PNS. These subtypes have characteristic physiological, pathological and pharmacological properties. The attention in developing nicotinic therapeutics has focused on generating agonists (including partial agonists) that activate nAChRs. Clinical targets for these potential nicotinic drugs include cognitive performance, neurodegenerative diseases, schizophrenia, anxiety and depression, Tourette's syntdrome, epilepsy, pain, smoking cessation, ulcerative colitis, and others (Cassels et al., 2005; Daly, 2005; Jensen et al., 2005). Varenicline, a cytisine analog and a partial agonist of α4β2 nAChRs, was approved in 2006 by U.S. Food and Drug Administration for use as a smoking cessation aid (Rollema et al., 2007). However, many nicotinic agonists, including varenicline, have undesirable side effects that eliminated or limited their clinic applications. For example, an initially very promising potential pain medicine, tebanicline (ABT-594), a nicotinic agonist, failed recently in Phase II clinic trails because of strong gastrointestinal adverse effects (Jain, 2004; Jensen et al., 2005). It is believed that the side effects of tebanicline are caused by its strong α3β4 nAChR agonist activity. Moreover, varenicline, the new smoking cessation aid, which is generally viewed as a safe drug, causes nausea in nearly 30% of the clinic trail participants. This side effect of varenicline is, presumably, also caused by its α3β4 nAChR agonist activity (Mihalak et al., 2006). Thus, there is clearly a need to develop new nicotinic therapeutics that have much better pharmacological profiles than those of the nicotinic agonist on the market or under the development.
Citation or identification of any reference in this section and other sections of this application is not an admission that such reference is prior art to the present invention. The content of each and every of the cited references are herein incorporated by reference in their entirety.